This proposal seeks continued support for exploration of structure and function of the ribosome actively engaged in protein synthesis, by means of cryo-electron microscopy and single-particle classification and reconstruction methods. The new direct electron detection camera our microscope is equipped with offers unprecedented opportunities to study the functional dynamics of translation at close to atomic resolution. Furthermore, a new capability we have implemented and further developed, time-resolved cryo-EM, allows us to investigate processes with reaction times in the tens and hundreds of milliseconds, which are reaction times of interest in the study of decoding, initiation and error check mechanisms. The aims of the proposal, in collaboration with leading labs in eubacterial translation, are threefold: (1) investigate the structural basis of a newly discovered retrospectiv error checking mechanism through which the ribosome terminates protein synthesis after an incorrect amino acid has been added to the polypeptide chain; (2) visualize a pre-initiation complex in two states of activation of IF2 at high resolution, to characterize the structural changes accompanying activation, and develop a model of the activation process. In a parallel study, we will perform time-resolved experiments in the range of tens to hundreds of millisecond, to follow the process of subunit joining during initiation. (3) By collecting large sigle particle datasets from complete translation systems, we will obtain an exhaustive inventory of ribosome in all phases of translation. Using a novel method of manifold embedding based on the similarity ordering of the data, in collaboration with Dr. Abbas Ourmazd, we will determine the continuum of conformational states during the work cycle, and the free energy landscape of the ribosome under various conditions. These data will allow us to gain unprecedented insights into the structural dynamics of one of the most complex molecular machines in the cell.